Outer organic layer and internal repair mechanism protects pteropod Limacina helicina from ocean acidification Victoria L Peck1, Geraint A Tarling1, Clara Manno1, Elizabeth M Harper2, Eithne Tynan3. 1. British Antarctic Survey, High Cross, Madingley Rd, Cambridge, CB5 8NP, UK 2. Department of Earth Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EQ, UK 3. Ocean and Earth Science, National Oceanography Centre Southampton, University of Southampton Waterfront Campus, European Way, Southampton SO14 3ZH, UK Scarred shells of polar pteropod Limacina helicina collected from the Greenland Sea in June 2012 reveal a history of damage, most likely failed predation, in earlier life stages. Evidence of shell fracture and subsequent re-growth is commonly observed in specimens recovered from the sub-Arctic and further afield. However, at one site within sea-ice on the Greenland shelf, shells that had been subject to mechanical damage were also found to exhibit considerable dissolution. It was evident that shell dissolution was localised to areas where the organic, periostracal sheet that covers the outer shell had been damaged at some earlier stage during the animal’s life. Where the periostracum remained intact, the shell appeared pristine with no sign of dissolution. Specimens which appeared to be pristine following collection were incubated for four days. Scarring of shells that received periostracal damage during collection only became evident in specimens that were incubated in waters undersaturated with respect to aragonite, Ar≤1. While the waters from which the damaged specimens were collected at the Greenland Sea sea-ice margin were not Ar ≤1, the water column did exhibit the lowest Ar values observed in the Greenland and Barents Seas, and was likely to have approachedAr≤1 during the winter months. We demonstrate that L. helicina shells are only susceptible to dissolution where both the periostracum has been breached and the aragonite beneath the breach is exposed to waters of Ar≤1. Exposure of multiple layers of aragonite in areas of deep dissolution indicate that, as with many molluscs, L. helicina is able to patch up dissolution damage to the shell by secreting additional aragonite 1 internally and maintain their shell. We conclude that, unless breached, the periostracum provides an effective shield for pteropod shells against dissolution in waters Ar≤1, and when dissolution does occur the animal has an effective means of self-repair. We suggest that future studies of pteropod shell condition are undertaken on specimens from which the periostracum has not been removed in preparation. Key words Limacina helicina Ocean acidification Periostracum Greenland Sea ice Pteropod 2 INTRODUCTION Since the start of the Industrial Revolution, about 48% of the anthropogenic CO2 emitted to the atmosphere has been sequestered into the world’s oceans (Sabine et al., 2004). This excess CO2 is dissolved into the surface ocean and reacts with seawater, causing pH and 2 − dissolved carbonate ion concentrations, [CO3 ], to fall, a phenomenon commonly referred to as ocean acidification (Caldeira & Wickett, 2003; Orr et al., 2005). In the polar-regions, ocean acidification is further exacerbated by increased solubility of gases within colder waters (Fabry et al., 2009) and also by sea ice processes, which can amplify seasonal variability in saturation state of mixed layer waters (Fransson et al., 2013). Furthermore, increased ice melt is freshening the mixed layer, lowering Total Alkalinity (TA) and Dissolved Inorganic Carbon (DIC) concentration. Carbonate saturation of polar waters is rapidly falling to values where aragonite, the less stable form of calcium carbonate (Mucci, 1983), becomes susceptible to dissolution (Ar ≤1; Orr et al., 2005). Pteropods, or ‘sea butterflies’, are pelagic gastropods which have evolved ‘wings’ derived from the foot enabling them to swim. The delicate shells of pteropods are made of the metastable aragonite and thus may be particularly prone to dissolution. The true polar pteropod, Limacina helicina, is a keystone species within polar ecosystems (Lalli & Gilmar 1989; Comeau et al., 2009; Hunt et al., 2008; Hunt et al., 2010). Living in the upper few hundred meters of the water column, in waters which are becoming increasingly undersaturated with respect to aragonite, L. helicina is frequently presented as being the “canary in the coal mine” for ocean acidification (e.g. Orr et al., 2005). Incubation experiments, investigating the response of the Arctic sub-species L. helicina helicina (Hunt et al., 2010) to elevated pCO2 scenarios indicate reduced net calcification (Comeau et al., 2010) and degradation in shell condition in undersaturated waters (Lischka et al., 2011). Observations of living specimens collected from a region of upwelling in the Southern Ocean, suggest that Antarctic sub-species L. helicina antarctica (Hunt et al., 2010) is subject to extensive dissolution where Ar =1 (Bednarsek et al., 2012a). However, many species of mollusc thrive with undersaturated waters, for example within freshwater or deep-sea hydrothermal vent communities, on account of their calcareous shells being protected from dissolution by the presence of a protective organic coating, a periostracum, covering their shells (Taylor & Kennedy, 1969; Harper, 1997). Possession of a periostracum is a shared 3 character for all shelled molluscs (Harper, 1997). This thin organic sheet of the periostracum is secreted at the edge of the mantle and is the first formed layer of the shell. The primary function of the periostracum is to separate the site of calcification from the ambient water and to provide the initial template onto which the shell is crystallised. It is this isolation of the extrapallial space by the periostracum that allows calcification to occur within waters which are undersaturated with respect to carbonate, with extreme examples being the occurrence of molluscs within hydrothermal vent (e.g. Tunnicliffe et al., 2009) and freshwater environments (e.g. Harper, 1997). A secondary function of the periostracum is to provide a protective veneer shielding the shell from the corrosive effects of undersaturated waters or chemical attack from predators (Harper, 1997). Of critical note, since the periostracum is only formed at the actively growing shell margin (Saleuddin & Petit 1983), thinning or loss of the periostracum via physical and biotic abrasion, epibiont erosion and bacterial decay will limit its effectiveness as protection as there is no possibility of repairing it once it is damaged. Although there has been very little published on biomineralization in pteropods, it is clear from the shell microstructure (Bandel, 1990) that they follow the typical molluscan pattern. In the case of Limacina the shell is composed of well ordered crossed-lamellar and prismatic aragonite layers, internal to an ultra-thin (<1 m) periostracum (Sato-Okoshi et al., 2010). The findings of Bednarsek et al. (2012a, 2012b) seem to suggest that pteropods receive little benefit from their periostracum when exposed to undersaturated waters. Quantification of pteropod shell loss by Bednarsek et al (2014a), found that 14 day exposure to undersaturated waters (Ar =0.8) resulted in a shell loss of 17.1%±3.0%. While the rate of dissolution reported by Bednarsek et al (2014a) is less than that predicted for the dissolution rate of pure aragonite, the fact that dissolution was reported over the entirety of the shells, questions the effectiveness of the periostracum for L. helicina antarctica. However, we note that Bednarsek et al. (Bednarsek et al., 2012a; Bednarsek et al., 2012c; Bednarsek et al., 2014b; Bednarsek et al., 2015) used chemical and plasma etching methods on shells prior to imaging, with the intention of removing the periostracum. Given the protective role of the periostracum in other shelled molluscs living in undersaturated waters, we opt to examine the relationship between dissolution and periostracal cover on specimens that are not subject to any preparation steps that would compromise the condition of the perioistracum or the shell beneath. Our minimal preparation approach intends to establish how effective the periostracum is in protecting the shells of pteropods. 4 Here we present our observations of L. helicina helicina shells collected from the Greenland and Barents Seas in June 2012 and the result of a small scale incubation experiment to assess the effectiveness of the periostracum, and therefore vulnerability, of this species to ocean acidification in the Arctic. METHODS This study carried out observations and incubations on L. helicina helicina specimens recovered during routine motion-compensated plankton net deployments during research cruise JR271 on board RRS James Clark Ross in June-July 2012. L. helcinia helicina specimens were recovered at three sites within the Greenland and Barents Seas as detailed in Table 1 and Figure 1. Water column structure and chemistry and manipulation of seawater for incubation Vertical CTD profiles were performed to characterise important water column structure (temperature, salinity, Chl-a) and carbonate chemistry. The depth of water collection for the experimental setup was then determined based on these initial profiles. The unfiltered water was collected from dedicated CTD casts and transferred to acid-cleaned clear 1 L Duran bottles and then sealed pending carbonate chemistry manipulation and the addition of pteropods. Subsamples at time zero were taken directly from the CTD